The present invention relates to a process for qualitatively and quantitatively analyzing a protein annexin-V which is present in a urine of a human and a mammalian animal (and which will-be referred to as annexin-V in a urine hereinafter), as well as a qualitatively and quantitatively analyzing regent and an application thereof.
The present invention also relates to a process for quantitatively analyzing annexin-V present in a urine using anti-annexin-V monoclonal antibody and polyclonal antibody having a specificity to an antigenic determinant side on a protein of a protein annexin-V, present in a human and a mammalian animal, thereby detecting the presence and absence of an organopathy of an internal organ such as kidney, heart and lung, which is combined with, for example, a disseminated intravascular coagulation syndrome (DIC) or a septicemia of a human by the resulting content of annexin-V in the urine, whereby particularly the disseminated intravascular coagulation syndrome (DIC) can be early diagnosed, and a diagnosis medicine for use in such analyzing process.
Further, the present invention relates to the measurement of annexin-V present in a urine of a human and a mammal such as a rat, which is capable of diagnosing the presence or absence of an organopathy of an internal organ by experimentally measuring a concentration of annexin-V in a urine of a rat, for example, measuring a concentration of annexin-V in a urine at the time when the blood annexin-V concentration has been risen, as well as the application or utilization thereof for the therapy of disseminated intravascular coagulation syndrome and for the development of an effective therapeutic medicine.
Yet further, the present invention relates to a process for quantitatively analyzing annexin-V present in a urine of a human and a mammal using an anti-annexin-V monoclonal antibody and polyclonal antibody having a specificity to antigenic determinant sides on a protein of a protein annexin-V in the human and the mammal, thereby enabling the diagnosis, particularly, of myocardial infarction and angina pectoris, based on the content of the annexin-V in the urine, and to a diagnosis medicine for use in such diagnosis.
The detection of the presence or absence of a disease of an internal organ with a substance present in a urine is less often than with a substance in blood or serum. The reason is that each of various substances in the urine and the value of pH of the urine exert an influence to a system to be measured.
As used in the present invention, the annexin-V which is an antigen is a protein having a molecular weight, for example, of 32 to 35 K dalton, i.e., a calcium-binding protein present in tissues and cells of a human and various mammals. The annexin-V is present in a soluble component of a cytoplasm and forms a family by an amino acid sequence. Presently, annexin I to XII are known. It is also known that the annexin can be bonded with phosphatide or actin depending on the concentration of calcium to exhibit anti-inflammatory effect and an anti-coagulation effect.
It has been reported that an annexin, which is one member of an annexin family, is contained in the lung, heart and kidney in the content increased in the named order, and is contained in a smaller content in the cerebrum and liver (see Heart Vessels, 1994, 9: 148-154). The present inventors have reported that the measurement of a concentration of annexin-V in blood is effective, for example, for the diagnosis of myocardial infarction, when the necrosis of tissues and cells has occurred, from the viewpoint that the annexin-V early departs into the blood (see Literature CCA, 1996), and has proposed a process for analyzing annexin-V present in blood using an anti-annexin-V antibody (see International Publication Number: WO96/15152 internationally published in May 23, 1996).
However, the measurement of a concentration of annexin-V in blood suffers from a problem that a pain is inflicted to a subject upon the drawing blood, and the concentration of annexin-V must be measured within a short period of time after the drawing blood, but much labor is required to separate serum from the drawn blood. Another problem is that EDTA must be added in order to control the bonding of various proteins contained in serum with calcium ion, and much labor is required for the preparation and addition of such a solution.
It is less often to detect the presence or absence of an organopathy of an internal organ with a substance present in a urine, than in a blood or serum, because a system to be measured is largely influenced by each of various substances contained in a urine and a larger value of pH of the urine. However, the present inventors have found from the subsequent researches and studies that the annexin-V in the urine stoichiometrically forms an antigen/antibody complex in a urine by an antigen-antibody reaction with an anti-annexin-V monoclonal antibody.
Further, the present inventors have found that the protein annexin-V in a human and a mammal is usually present only in an amount of about 2 ng/ml in a urine in a healthy person, but when a person has an organopathy in an internal organ, wherein the content of annexin-V departing from the internal organ is larger, the annexin-V appearing in his or her blood also appears immediately in his urine.
Yet further, from the viewpoint that if a concentration of annexin-V appearing in a urine can be measured quantitatively, the presence or absence of an organopathy can be diagnosed, the present inventors have found by continuing the measurement of the concentration of annexin-V in the urine that when it is observed, by continuing the measurement of the concentration of annexin-V in the urine, that the rising of the concentration of annexin-V in the urine has occurred, for example, due to an internal organ disorder or the destruction of tissues of kidney caused by a blood circulation failure in a capillary vessel, the amount of annexin-V in the urine is increased, and that the degree of a disorder of an internal organ such as lung, heart and kidney and the therapy and recuperation of such disorder can be diagnosed early, based on an increase or decrease in concentration of the annexin-V in the urine. Moreover, the present inventors have found that the rising of the concentration of annexin-V is observed in a urine in acute nephritis, but not observed in blood.
It is an object of the present invention to provide a process for analyzing annexin-V, wherein the measurement of annexin-V can be carried out easily without need for the addition of a regent for inhibiting the bonding of various proteins with calcium ion and for regulating a specimen solution, and a process for diagnosing an internal organ disorder or the like, based on such analyzing process.
The present invention resides in a process for analyzing annexin-V in a urine, comprising steps of bringing a urine into contact with an anti-annexin-V monoclonal antibody to perform an antigen-antibody reaction of annexin-V present in the urine with the anti-annexin-V monoclonal antibody, thereby forming an annexin-V antigen/anti-annexin-V monoclonal antibody complex, and quantitatively measuring the amount of the formed annexin-V antigen/anti-annexin-V monoclonal antibody complex. The present invention also resides in a process for analyzing annexin-V in a urine, comprising the steps of bringing a urine into contact with a first anti-annexin-V monoclonal antibody fixed in a solid phase to perform an antigen-antibody reaction of annexin-V present in the urine with the first anti-annexin-V monoclonal antibody, thereby forming an annexin-V antigen/first anti-annexin-V monoclonal antibody complex; bonding an anti-dog-annexin-V polyclonal labeled antibody or a second anti-annexin-V monoclonal labeled antibody to the annexin-V antigen of the formed antibody complex to form a bonded antibody complex/anti-annexin-V polyclonal or second anti-annexin-V monoclonal labeled antibody product; and quantitatively measuring the amount of the formed labeled antibody bonded product. Further, the present invention resides in a regent for analyzing annexin-V in a urine, comprising a first anti-human-annexin-V monoclonal antibody having a specificity to an antigen determinant site on the protein of a human annexin-V antigen fixed in a solid phase, and a second anti-human-annexin-V monoclonal labeled antibody or an anti-dog-annexin-V polyclonal labeled antibody having a binding specificity to the antigen determinant site on the protein of the human annexin-V antigen.
In addition, the present invention is directed to a process for diagnosing an organopathy, comprising the steps of measuring a concentration of annexin-V in a urine, and comparing the measured value of the concentration of the annexin-V in the urine with a normal value of concentration of annexin-V. Further, the present invention is directed to a process for diagnosing an organopathy by use of a urine, comprising the steps of bringing a urine into contact with a first anti-annexin-V monoclonal antibody to perform an antigen/antibody reaction of annexin-V in the urine with the first anti-annexin-V monoclonal antibody, thereby forming an annexin-V antibody/first anti-annexin-V monoclonal antibody complex; bonding an anti-dog-annexin-V polyclonal labeled antibody or a second anti-annexin-V monoclonal labeled antibody to the annexin-V antigen of the formed antibody complex to form a bonded product of anti-dog-annexin-V polyclonal labeled antibody or second anti-annexin-V monoclonal labeled antibody/annexin-V antigen/antibody complex; and quantitatively measuring the amount of the formed bonded product of anti-dog-annexin-V polyclonal labeled antibody or second anti-annexin-V monoclonal labeled antibody/annexin-V antigen/antibody complex. Yet further, the present invention is directed to a medicine for diagnosing an organopathy by using a urine as specimen, comprising a first anti-human-annexin-V monoclonal antibody having a binding specificity to an antigen determinant site on the protein of a human annexin-V antigen fixed in a solid phase, and a second anti-human-annexin-V monoclonal labeled antibody or an anti-dog-annexin-V polyclonal labeled antibody having a binding specificity to the antigen determinant site on the protein of the human annexin-V antigen.
Yet further, the present invention is directed to a process for diagnosing acute nephritis by use of a urine, comprising the steps of bringing samples of urine taken at two different time points into contact with an anti-annexin-V monoclonal antibody to perform an antigen/antibody reaction of annexin-V in each of the urine samples with the anti-annexin-V monoclonal antibody, thereby forming an annexin-V antigen/anti-annexin-V monoclonal antibody complex; quantitatively measuring the amount of the formed annexin-V antigen/anti-annexin-V monoclonal antibody complex, thereby determining a concentration of annexin-V in each of the urine samples to determine a difference between the concentrations of annexin-V in the urine samples taken at the two different time points from the values of the concentrations of annexin-V in the urine samples taken at the two different time points; and on the other hand, bringing annexin-V present in a specimen derived from samples of blood drawn at the two different time points into an antigen/antibody reaction with an anti-annexin-V monoclonal antibody to form an annexin-V antigen/anti-annexin-V monoclonal antibody complex; quantitatively measuring the amount of the formed annexin-V antigen/anti-annexin-V monoclonal antibody complex, thereby determining a concentration of annexin-V in each of the blood samples to determine a difference between the concentrations of annexin-V in the blood samples drawn at the two different time points; and comparing the differences between the annexin-V concentrations in the urine samples and the blood samples taken at the two different time points with each other. Additionally, the present invention is directed to a process for diagnosing acute nephritis by use of a urine, comprising the steps of bringing samples of urine taken at two different time points into contact with an anti-annexin-V monoclonal antibody to perform an antigen/antibody reaction of annexin-V in each of the urine samples with the anti-annexin-V monoclonal antibody, thereby forming an annexin-V antibody/anti-annexin-V monoclonal antibody complex; quantitatively measuring the amount of the formed annexin-V antibody/anti-annexin-V monoclonal antibody complex, thereby determining a concentration of annexin-V in each of the urine samples to determine a difference between the concentrations of annexin-V in the urine samples taken at the two different time points from the values of the concentrations of annexin-V in the urine samples at the two different time points; and on the other hand, bringing annexin-V in a specimen derived from samples of blood drawn at the two different time points into an antigen/antibody reaction with an anti-annexin-V monoclonal antibody, thereby forming an annexin-V antigen/anti-annexin-V monoclonal antibody complex; quantitatively measuring the amount of the formed annexin-V antigen/anti-annexin-V monoclonal antibody complex, thereby determining a concentration of annexin-V in each of the blood samples to determine a difference between the concentrations of annexin-V in the blood samples at the two different time points; and comparing the differences between the concentrations of annexin-V in the urine samples and the blood samples taken at the two different time points with each other. Further, the present invention is directed to a medicine for diagnosing acute nephritis by use of a urine, comprising a regent for analyzing annexin-V present in a urine, and a regent for analyzing annexin-V present in blood.